Design,Synthesis And Biological Activity Evaluations Of Novel Immunodulatory Drugs（IMiDs）

Multiple myeloma（MM）is a disease with malignant proliferation of plasma cells.Typical clinical manifestations:hemorrhage,bone pain,renal dysfunction and abnormal immune function,etc.In recent years,some new drugs and targeted therapy have improved the overall prognosis of MM patients,but very few patients can achieve long-term complete remission,and most patients will still relapse after treatment,develop drug resistance,and eventually die of the disease.With the continuous research on MM immunotherapy,immunomodulatory drugs（IMi Ds）represented by thalidomide have been put into clinical use.The prognosis of the disease was improved to a great extent and the survival time of the patients was prolonged significantly.Cereblon（CRBN）is a protein involved in the construction of E3 ubiquitin ligase,which can be degraded by the proteasome by ubiquitinating substrate proteins.CRBN expression is associated with the proliferation inhibition activity of IMi Ds and is a potent therapeutic target in MM cells.The interaction of IMi Ds with CRBN can degrade Ikaros（IKZF1）and Aiolos（IKZF3）proteins,while down-regulating downstream target proteins interferon regulatory factor 4（IRF4）and MYC（c-MYC）,it is transmitted downstream through the two signaling pathways of IRF4 and c-MYC to play a tumor-killing role.In this paper,a series of compounds containing isoquinoline-1,3（2H,4H）-diketone fragments were designed and synthesized on the basis of the drug structures of IMi Ds on the market,and their synthesis methods,structure-activity relationships and biological activities were studied.Two routes have been designed for the synthesis of the compounds.The first synthetic route uses the perphthalic anhydride derivative as the starting material,and cyclizes with 3-aminopiperidine-2,6-dione hydrochloride to obtain compound（1-3）;the second synthetic route uses the o-chlorophenylacetic acid derivative as the starting material,through substitution,amidation,hydrolysis,cyclization and reduction reactions to obtain compound（1-10）.Taking the human myeloma cell line NCI-H929 as the research object,the biological activity of the synthesized compounds was studied.The results of the CCK8 experiment showed that all the 10 synthesized compounds exhibited good proliferation inhibitory activity on the MM cell line NCI-H929.Among them,the proliferation inhibitory activity of compound 5 was stronger(IC₅₀=2.25μmol/L),which was comparable to that of lenalidomide(IC₅₀=1.12μmol/L).Flow cytometry apoptosis experiments showed that compound 5 could induce apoptosis of NCI-H929cells.Compound 5 with drug concentrations of 0.0μM,0.1μM,0.5μM,1.0μM,and5.0μM treated NCI-H929 cells for 72 h,the apoptosis rates were 6.0%,13.4%,21.5%,29.5%,and 34.6%,respectively.The apoptosis effect was time and dose dependent.The study on the distribution of NCI-H929 cell cycle showed that with the increase of drug concentration,the ratio of NCI-H929 cells in G0/G1 phase increased,proving that the effect of compound 5 on NCI-H929 cell cycle was mainly G0/G1 phase arrest.On the Auto Dock 4.2.6 platform,the docking structure and binding position of lenalidomide,compound 5 and CRBN-DDB1 protein were verified virtually.Molecular docking results showed that compound 5 could effectively dock with CRBN protein,confirming that CRBN protein is a potential target of compound 5 in the treatment of MM.The results of Western blot experiment showed that when the concentration of compound 5 was 10μmol/L,the expression of IKZF3 protein was significantly decreased,and the expression of IKZF1 protein was completely disappeared,and compound 5 regulated the expression of these two proteins in a dose-dependent manner.The results demonstrate that compound 5 leads to the ubiquitination degradation of IKZF1 and IKZF3 proteins through the interaction with CRBN.In vitro bioactivity experiments proved that the compound 5 designed and synthesized in this paper exhibited biological activity comparable to that of lenalidomide,it has good tumor growth inhibition,apoptosis induction and cell cycle arrest ability,which could be used as a new type of IMi Ds for the treatment of MM.At the same time,compound 5 can degrade the ubiquitination of disease-related proteins by inducing CRBN,and has the potential to construct proteolysis-targeting chimeras（PROTACs）.